The present invention pertains to polynucleotides derived from previously unknown sequences internal to the ddl gene coding for D-Alanine:D-Alanine ligase of various bacterial strains belonging to the Enterococci or to the Streptococci genus. This invention also relates to the use of the polynucleotides as oligonucleotide primers or probes for detecting and identifying specifically Enterococci or Streptococci in a biological sample.
In another embodiment, the present invention is directed to the full length coding sequences of the ddl genes from Streptococci and Enterococci and to the polypeptides expressed by these full length coding sequences.
Further, this invention relates to the use of the expressed polypeptides to produce specific monoclonal or polyclonal antibodies that serve as detection means in order to characterize a specific species or group of species of Streptococci or Enterococci.
The present invention is also directed to diagnostic methods for detecting specifically strains of Enterococci or streptococci expected to be contained in a biological sample. The diagnostic methods use the oligonucleotide probes or primers as well as the antibodies of the invention.
Glycopeptide antibiotics, such as vancomycin and teicoplanin, inhibit peptidoglycan synthesis in Enterococci and are used to treat severe infections caused by Gram-positive bacteria. Glycopeptides do not interact directly with cell wall biosynthetic enzymes, but form complexes with peptidoglycan precursors and prevent their incorporation into the cell wall. Consequently, the activity of glycopeptides is determined by the substrate specificity of the enzymes that affect the structure of peptidoglycan precursors.
Acquisition of glycopeptide resistance results from the transfer of mobile genetic elements that encode enzymes for the synthesis of low-affinity precursors and elimination of the high-affinity precursors normally produced by the host. Among the enzymes of altered substrate specificity found in glycopeptide resistant Enterococci strains, D-Ala:D-Ala ligase is a main component. More precisely, usually, the D-Ala:D-Ala related enzymes synthesize D-alanyl-D-Alanine. In vancomycin-resistant Gram-positive bacteria, structurally related enzymes synthesize D-Alanyl:D-Lactate or D-Alanyl:D-Serine.
PCT Application No. PCT/FR91/00855 (Courvalin et al.), published on May 14, 1992, discloses a nucleotide sequence from the Enterococcus faecium strain BM4147, the Van A gene, that encodes a D-Ala:D-Lac ligase of wide specificity. D-Ala:D-Lac ligase is involved in the resistance of this bacterial strain to the glycopeptide antibiotics vancomycin and teicoplanin. This PCT Application discloses a pair of degenerate primers, V1 and V2, that can be used as primers or probes in order to detect, under non-stringent hybridization conditions, Enterococcus strains that exhibit resistance to glycopeptide antibiotics. Nevertheless, there continues to exist a need in the art for polynucleotides specific for bacterial strains of the Streptococci and Enterococcus genuses.